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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyLeber congenital amaurosis (LCA). Recently, a dominant mutationD477G of RPE65 has been reported to cause RP through themechanism yet to be identified. The purpose of this study is to revealhow the mutation D477G of RPE65 affects its enzymatic activity.Methods: Recombinant human wt RPE65 (hRPE65) with a His-tagand its mutant D477G with a 1D4 tag were generated by site-directedmutagenesis. Wt hRPE65 and the mutant were expressed in 293-LRAT cells using either adenovirus or plasmid transfection and theirexpression was analyzed by Western blot analysis. Subcellularfractionation was performed using FractPrepTM kit. Catalyticactivities of wt hRPE65 and the mutant were quantified by HPLC.Velocity sedimentation through a sucrose gradient was used toidentify oligomer formation between the mutant and wt RPE65.Results: Expression level of the D477G mutant was found similar tothat of wt hRPE65. The wt hRPE65 showed a high stability, with ahalf-life more than 10 h whereas the mutant has a decreased half-life,suggesting that the mutation decreases the protein stability. Themutant showed a diminished catalytic activity in comparison to thatof wt hRPE65. The subcellular fractionation revealed thatconsiderable amount of wt and D477G were located in membranefractions. However, when wt and D477G were co-expressed, itcaused a decrease of wt RPE65 in the membrane fraction while asimultaneous increase in its inclusion body fraction. Moreover, coexpressionof the D477G mutant with wt RPE65 enhances the RPE65aggregation and causes oligomerization of the wt and the mutantRPE65.Conclusions: The presence of the D477G mutant affects membraneassociation of wt RPE65. The D477G mutation enhances theformation of RPE65 oligomers rather than dimers or monomers. Inturn, the oligomerization affects the isomerase activity of wt RPE65and leads to impaired vision in patients with the D477G mutation ofRPE65.Commercial Relationships: Olga Nikolaeva, None; Gennadiy P.Moiseyev, Charlesson LLC (E); Yusuke Takahashi, None; Jian-Xing Ma, NoneSupport: NIH EY018659, EY012231, EY019309 andP20GM104934Program Number: 3763 Poster Board Number: A0102Presentation Time: 2:45 PM - 4:30 PMHuman iPS-RPE Synthesize and Release 11-cis Retinaldehydefrom Exogenous All-trans RetinolAlberto Muniz 1, 2 , Mark L. Plamper 2 , Jae Hyek Choi 1, 2 , Whitney A.Greene 1, 2 , Anthony J. Johnson 1 , Andrew T. Tsin 3 , Heuy-Ching H.Wang 1 . 1 Ocular Trauma, United States Army Institute of SurgicalResearch, Fort Sam Houston, TX; 2 National Research Council,Washington, DC; 3 Biology, The University of Texas at San Antonio,San Antonio, TX.Purpose: Retinal pigment epithelium (RPE) has recently beenderived from human induced pluripotent stem (iPS) cells. Beforenewly derived iPS-RPE cells can be used for stem cell therapies, it isimportant to determine the functional ability of iPS-RPE. One crucialrole of the RPE is uptake and processing of retinoids to supplyphotoreceptors with visual chromophore. However, the ability of iPS-RPE to support visual pigment regeneration has not beendemonstrated. The purpose of this study is to investigate the retinoidprocessing ability of the visual cycle in cultured iPS-RPE.Methods: RPE derived from human iPS cells were analyzed by RT-PCR and western blot analyses to detect expression of RPE visualcycle genes and proteins LRAT, RPE65, CRALBP and the RPEspecific gene PEDF. To examine the retinoid processing ability of theiPS-RPE, all-trans retinol (all-trans ROL) was delivered to culturediPS-RPE. After a 24 hour incubation period, retinoids were extractedfrom cell homogenates and the culture media. The extracts wereanalyzed by HPLC on a 0.2% to 10% dioxane/hexane gradientsystem. Retinoids were identified by retention time and respectiveabsorbance spectrum.Results: Expression of the critical visual cycle components LRAT,RPE65, CRALBP and the RPE specific gene PEDF was confirmedby RT-PCR and western blot in iPS-RPE. In addition, followingincubation with all-trans ROL, all-trans retinyl palmitate wasdetected from cell homogenates of iPS-RPE by HPLC . Furthermore,HPLC analysis of the culture media revealed a peak corresponding tothe retention time and absorbance spectrum of 11-cis retinaldehyde(11-cis RAL). Retinoids were not detected in control samples.Conclusions: iPS-RPE maintains expression of visual cycle proteins.The detection of all-trans retinyl palmitate in iPS-RPE and 11-cisRAL in the culture media demonstrates that the iPS-RPE can uptake,process, and secrete retinoids, confirming that the visual cycleproteins expressed in iPS-RPE are functional and can support visualpigment regeneration. This is the first study to demonstrateexpression of visual cycle machinery and complete visual cycleactivity in stem cell-derived RPE.Commercial Relationships: Alberto Muniz, None; Mark L.Plamper, None; Jae Hyek Choi, None; Whitney A. Greene, None;Anthony J. Johnson, None; Andrew T. Tsin, None; Heuy-ChingH. Wang, NoneSupport: US Army Medical Research Command Clinical andRehabilitative Medicine Research Program.Program Number: 3764 Poster Board Number: A0103Presentation Time: 2:45 PM - 4:30 PMFunctional Characterization of Peropsin in the Retinal PigmentEpitheliumJeremy D. Cook, Roxana A. Radu, Hui Sun, Gabriel H. Travis.Ophthalmology, Jules Stein Eye Institute, UCLA School ofMedicine, Los Angeles, CA.Purpose: Peropsin (RRH) is a non-visual opsin of unknown functionlocated in apical processes of the retinal pigment epithelium (RPE).The current study is an attempt to determine the function of peropsin.We began with the hypothesis that peropsin is involved in theprocessing of visual retinoids. To this end, we compared levels ofvisual retinoids in wild-type and peropsin-knockout (rrh-/-) miceunder dark-adapted conditions, and at different times following adeep photobleach. We are employing a cell-culture system to studythe role of peropsin on retinoid processing by enzymes of the visualcycle in RPE cells.Methods: Mice homozygous for a null mutation in the rrh gene weregenerated using conventional gene-targeting methods. Both the rrh-/-and strain-129 control mice (WT) were homozygous for the L450(normal) allele of rpe65. Mice were dark-adapted overnight. A subsetof WT and rrh-/- mice were euthanized and their eyes collected,while the remainder were exposed to fluorescent light at 1,000 lux for5 minutes. Subsets of these mice were euthanized immediatelyfollowing light exposure, or returned to darkness for different timeperiods before euthanasia. Retinas and RPE-containing eyecupsminus retinas were dissected, homogenized, and extracted withhexane for HPLC analysis of retinoids. To assay peropsin expressionand function in cultured HEK-293T cells, a construct containing thegene for mouse peropsin was subcloned into pcDNA and used fortransient transfection. Expression was confirmed by immunoblotanalysis and confocal microscopy, retinoid uptake was examined byHPLC.Results: Levels of all-trans-retinyl esters were several-fold lower inRPE from rrh-/- versus WT mice under all light conditions. Fiveminutes following the photobleach, levels of 11-cis-retinaldehyde and©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biology11-cis-retinyl esters were higher in the RPE of rrh-/- versus WTmice. Levels of LRAT, Rpe65, RGR-opsin and LRAT were similarin RPE from the two strains. Peropsin immunoreactivity wasobserved in the plasma membranes of 293T cells following transienttransfection with the peropsin expression plasmid.Conclusions: The altered retinoid dynamics in rrh-/- mice suggestdelayed removal of 11-cis-retinaldehyde from RPE and delayeduptake of all-trans-retinol by RPE from bleached photoreceptors.Peropsin-transfected HEK-293T cells represent a suitable system tostudy the role of peropsin on the uptake and processing of visualretinoids.Commercial Relationships: Jeremy D. Cook, None; Roxana A.Radu, None; Hui Sun, None; Gabriel H. Travis, NoneSupport: 5R01 EY011713Program Number: 3765 Poster Board Number: A0104Presentation Time: 2:45 PM - 4:30 PMInterphotoreceptor Retinoid Binding Protein Protects andDelivers Retinoids in the Cone Visual CycleAndrew T. Tsin 1 , Joshua M. Mimun 1 , Federico Gonzalez-Fernandez 2 .1 Biology, University of Texas San Antonio, San Antonio, TX;2 Veterans Affairs and SUNY Eye Institute, State University of NewYork at Buffalo, Buffalo, NY.Purpose: In the cone cycle, the recycling of all-trans retinol (atROL)and 11-cis-retinol (11cROL), between Müller cells and conephotoreceptor cells occurs within the retina. Interphotoreceptorretinoid binding protein (IRBP), the most abundant soluble protein inthe interphotoreceptor matrix, can bind to these photosensitiveretinoids, suggesting a role for IRBP as a transfer protein in the conevisual cycle. Thus, we hypothesized that IRBP is able to protectatROL and 11cROL from light induced degradation anddeliver/retrieve the retinoids to/from Müller cells.Methods: Protection studies: atROL and 11cROL (3.0 µM each)were exposed to 2,000 Lux, in the presence and absence of 3.0 µMIRBP. Retinoids were extracted every 10 min for 30 min andanalyzed by HPLC. Delivery/release studies: Primary chicken Müllercells (CMCs) were incubated for 24 hrs in serum free media (SFM)with 2.0 µM atROL and 2.0 µM IRBP or 2.0 µM BSA. To test ifthese proteins facilitated the release of retinoids from CMCs after aninitial 24 hr incubation with atROL and BSA (2.0 µM each) in SFM,the CMCs were re-incubated with SFM, 2.0 µM IRBP in SFM, or 2.0µM BSA in SFM for an additional 24hrs. Cellular homogenate andmedia were extracted for retinoids and analyzed via HPLC.Results: 30 min of light exposure resulted in degradation of 11cROL(83%) and atROL (67%). IRBP reduced these levels to 21%(11cROL) and 18% (atROL). After incubation with atROL for 24 hrs,the CMCs contained 0.32 pm/mg cell protein(cp) of all-trans retinylpalmitate (atRP) when incubated without proteins, 6.3 pm/mg cp ofatRP when incubated with BSA, and 0.85 pm/mg cp of atRP whenincubated with IRBP. The addition of BSA or IRBP into culturemedia of CMCs pre-incubated with atROL resulted in a significantincrease of atROL when compared to SFM (BSA: 31.7 pm/mg cp;IRBP: 40.1 pm/mg cp; SFM: 0.56 pm/mg cp).Conclusions: IRBP protects retinoids in situ from light-induceddegradation. IRBP delivery and retrieval experiments suggest thatIRBP delivers retinoids to the CMCs as indicated by the increase ofatRP present in the cellular homogenate and retrieves retinoids fromthe CMCs as indicated by the increase of atROL present in theculture media. IRBP could act as a transport protein of the conevisual cycle by protecting and facilitating transport of retinols toMüller cells.Commercial Relationships: Andrew T. Tsin, None; Joshua M.Mimun, None; Federico Gonzalez-Fernandez, PathRD Inc. (S)Support: ), NIH SCORE Program Grant # GM08194; NationalCenter for Research Resources (5G12RR013646-12); NationalInstitutes of Minority Health and Health Disparities(G12MD007591); Merit Review Award I01BX007080; NIH RO1EY09412Program Number: 3766 Poster Board Number: A0105Presentation Time: 2:45 PM - 4:30 PMBiochemical Characterization of Retinoid Isomerases in the RPEand RetinaQuan Yuan, Joanna J. Kaylor, Shanta Sarfare, Tongzhou Xu, JacobMakshanoff, Pengfei Cheng, Gabriel H. Travis. Jules Stein EyeInstitute, UCLA, Los Angeles, CA.Purpose: Enzyme mediated conversion of all-trans-retinoids to 11-cis-retinoids plays a key role in the biochemical process thatregenerates visual pigment for photoreceptor cells. RPE65, a knownprotein in RPE cell, has been confirmed as the isomerase in retinalpigment epithelium cells. Recently we identified dihydroceramidedesaturase 1 (DES1) as the retinoid isomerase for regenerating coneopsins in daylight. However the biochemical mechanism of theenzyme still remains elusive. Here we characterize the biochemicalproperties of DES1 using a variety of biochemical and biophysicalmethods.Methods: GST fused DES1 and His-CRALBP protein were purifiedby chromatography. Pull-down assays were carried out with thepurified proteins. The isomerase activities of RPE65 and DES1 incorresponding animal tissues and cell expression in the presence of avariety of specific inhibitors have been examined.Results: The physiological and reciprocal pull-down assays confirmthe direct interaction between DES1 and CRALBP. DES1 isomeraseactivity is dependent on the cytochrome b5 electron transfer chain.Inhibition of DES1 isomerase activity by spin traps and probesdemonstrate a possible radical intermediate pathway. DES1 is aFe(III) dependent retinol isomerase.Conclusions: Similarity towards different inhibitors for chickenretina and HEK-DES1 suggests DES1 is the isomerase-2 responsiblefor the alternative visual cycle in retina. The reaction mechanism ofDES1 appears to involve a free-radical intermediate.Commercial Relationships: Quan Yuan, None; Joanna J. Kaylor,None; Shanta Sarfare, None; Tongzhou Xu, None; JacobMakshanoff, None; Pengfei Cheng, None; Gabriel H. Travis, NoneSupport: 5R01 EY011713Program Number: 3767 Poster Board Number: A0106Presentation Time: 2:45 PM - 4:30 PMIsomerase 2 Activity in Primary Müller Cells from conedominatedChicken Retinas is iron-dependentBrandi S. Betts-Obregon, Andrew S. Mendiola, Andrew T. Tsin.Biology, The University of Texas at San Antonio, San Antonio, TX.Purpose: Previous studies have shown that there are two parallelvisual cycles in the cone dominated chicken retina (Muniz et al2009). Takahashi et al 2011 showed that RPE65c is an irondependentIsomerase 2 in the retinal Müller cells of the conedominant zebrafish. Kaylor et al 2012 showed that DES1 is anIsomerase 2 in the Müller glial cells of the retina. This DES1 proteinhas ion-binding di-iron ligands. We hypothesize that Müller cellslocated in the chicken retina have an iron-dependent IsomeraseMethods: Primary Müller cells were harvested from chicken retinasand seeded into T75 flasks. Cells were grown until confluence(approx 10-14 days) and homogenates prepared. Homogenates werepre-incubated with or without 5mM of 2,2’-Bipyridine (a knownisomerase 2 inbhibitor) for 30 minutes at room temperature.Homogenates were then incubated with 20 µM all-trans retinol©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
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